BackgroundLeeches (Hirudinida) play a significant role as intermediate hosts in the circulation of trematodes in the aquatic environment. However, the species richness, molecular diversity and phylogeny of larval stages of Strigeidae trematodes (tetracotyle) occurring in this group of aquatic invertebrates remain poorly understood. In the present work, on the basis of recently obtained sequences of several molecular markers we analysed several aspects of the ecology, taxonomy and phylogeny of the genera Australapatemon and Cotylurus, which utilise leeches as intermediate hosts.MethodsFrom April 2017 to September 2018, 153 leeches were collected from several sampling stations in small rivers with slow-flowing waters and related drainage canals located in three regions of Poland. The distinctive forms of tetracotyle metacercariae collected from leeches supplemented with adult Strigeidae specimens sampled from a wide range of water birds were analysed using the 28S rDNA partial gene, ITS2 region and the COI fragment.ResultsAmong investigated leeches, metacercariae of the tetracotyle type were detected in the parenchyma and musculature of 62 specimens (prevalence 40.5%) with a mean intensity reaching 19.9 ind. The taxonomic generic affiliation of metacercariae derived from leeches revealed the occurrence of two Strigeidae genera: Australapatemon Sudarikov, 1959 and Cotylurus Szidat, 1928. Phylogenetic reconstructions based on the partial 28S rRNA gene, ITS2 region and partial COI gene confirmed the separation of the Australapatemon and Cotylurus clades. Unfortunately, regarding currently available molecular data and our results, it is not possible to precisely define the taxonomic position of the recently sequenced tetracotyle of Australapatemon. On the other hand, on the basis of the obtained sequences, supplemented with previously published data, the metacercariae of Cotylurus detected in leeches were identified as two species: C. strigeoides Dubois, 1958 and C. syrius Dubois, 1934. This is the first record of C. syrius from the intermediate host.ConclusionsThe results suggest the separation of ecological niches and life cycles between C. cornutus (Rudolphi, 1808) and C. strigeoides/C. syrius with potential serious evolutionary consequences for a wide range of host–parasite relationships. Moreover, phylogenetic analyses corroborated the polyphyletic character of C. syrius, the unclear status of C. cornutus and the separate position of Cotylurs raabei Bezubik, 1958 within Cotylurus. The data demonstrate the inconsistent and confusing taxonomic status of the sequenced tetracotyle of Australapatemon, resulting, in our opinion, from the limited availability of fully reliable, comparative sequences of related taxa in GenBank.